1. Field of the Invention
This invention relates to a drying apparatus and method for DNA sequencing. More specifically, the present invention is directed to a drying apparatus and method for drying a gel that has undergone gel electrophoresis and before the DNA bands are visualized by autoradiography. Gel drying is a necessary step in for accurate DNA sequencing. The presence of water will adversely effect accurate determination of the DNA bands by autoradiography for two primary reasons. First, the film used in autoradiography will tend to stick to water in the gel, thereby preventing the exposed film from being separated from the gel, which in turn prevents the exposed film from being able to be developed. In addition, water can be a .beta. particle blocker, and thus, the presence of water in the gel will result in adverse readings.
The present invention includes the use of an apparatus comprising a turbulent-flow, gas moisture removing medium impingement-based subassembly, i.e. air impingement gel dryer. In the present invention, gel drying is accomplished by taking a gel that is adhered to glass plate directly from the electrophoresis device after electrophoresis and placing the gel and glass plate into the air impingement gel dryer.
Particular features of the present invention include the elimination of transferring the gel from a glass plate to paper for drying by a conventional vacuum gel dryer, decreasing the amount of time required using conventional oven drying of electrophoresis gel on a glass plate, and decreasing the amount of heat needed for sufficient gel drying.
These advantageous features of the present invention result in improved speed, reliability and readability of DNA sequence analysis. More specifically, the present invention results in reducing the time of gel drying by about one-third, and an improvement or at least 10% in the amount of DNA sequence that can be determined.
2. Description of the Prior Art
Gel electrophoresis is a fundamental biochemical separation technique that forms the basis for distinguishing a variety of biologically important molecules on the basis of size, charge or a combination thereof. Specific examples of biological molecules advantageously separated by gel electrophoresis include proteins and nucleic aids. Electrophoresis is usually performed in a gelled (e.g., agarose) or polymerized (e.g., polyacrylamide) media (generically termed a "gel") that contains an electrically conducting buffer. Electrophoresis is performed wherein a voltage is applied via chemically inert metal electrodes across the cross-sectional area of the gel. The biological sample of interest is placed into pre-formed sample wells in the gel, usually at one end of the gel, and the polarity of the applied voltage is arranged so that the biological sample migrates through the gel towards one of the electrodes (usually positioned at the opposite end of the gel from the samples). Where appropriate, the inverse linear relationship between migration distance and molecular size is maintained by the addition of chemical denaturants (such as urea, formamide, or sodium dodecyl sulfate) to the gel and electrophoresis buffer.
A particular application of gel electrophoresis is the separation of single-stranded DNA fragments in the determination of the nucleotide sequence of a nucleic acid of interest. To this end, a collection of single-stranded DNA fragments is generated either by chemical degradation of the nucleic acid (using the Gilbert method, see e.g., Maxam and Gilbert (1980), Methods Enzyme, 65, p499-500) or by replacement DNA synthesis using a polymerase (using the Sanger method, see e.g., Sanger, F., Niklen, S., and Coulson, A. R. (1977) Proc. Nat. Acad. Sci. USA 74, p5463-5467). This collection of single stranded DNA fragments includes a fragment corresponding to each position in the sequence to be determined; in the most frequently-used sequencing method, this correspondence is directly related to the distance from a fixed site of initiation of polymerization at a primer that is annealed to the nucleic acid to be sequenced. Thus, determination of the desired sequence depends on the separation of each of the fragments, which differ in length by only a single nucleotide.
Traditionally, the identity of each of the possible nucleotides at each position (adenine, guanine, cytosine or thymidine) is distinguished by performing a sequencing reaction specific for each ending nucleotide in separate chemical reaction mixtures. Thus, each sequencing experiment is typically performed in 4 separate tubes, wherein are generated a collection of fragments each ending at a position corresponding to the terminating nucleotide used in that reaction. A nucleotide sequence is thereafter determined by performing denaturing gel electrophoresis on each of a set of 4 reactions, each reaction electrophoresed individually in adjacent lanes of a single sequencing gel. The presence of a band at a position in a nucleotide-specific lane of such a gel indicates the identity of that nucleotide at that position in the sequence. Using conventional techniques, each of the fragments is radiolabeled, and the bands are visualized after electrophoresis by autoradiography.
A number of constraints limit the extent of nucleotide sequence information that can be obtained when conventional gel drying of electrophoresis gels is used. The primary constraint is the amount of diffusion of DNA sequence bands. More specifically, this diffusion, also called band broadening, on the autoradiographic film results in diminished DNA sequence readings and determination. Moreover, the transfer of the gel from the glass plate to the paper can physically distort the gel sufficiently enough to cause band broadening.
Diffusion of DNA sequence bands that results from the use of conventional gel drying consists of a number of diffusion factors, which include, temperature and time factors. The greater the temperature and time needed to dry the gel, the greater the Brownian motion of the DNA molecules and the greater the diffusion of DNA sequence bands. When the gel is transferred from a glass plate to paper for drying by a conventional vacuum gel dryer, other diffusion factors arise, including diffusion due to the fact that paper has far greater porosity than glass.
The total variance of a peak in field-amplified capillary electrophoresis has been defined by the following equation: EQU .sigma..sup.2 =.sigma..sub.diff.sup.2 +.sigma..sub.inj.sup.2 +.sigma..sub.ther.sup.2 +.sigma..sub.a.sup.2
where .sigma..sub.diff.sup.2 is the variance due to diffusion (which in turn varies with temperature and time), .sigma..sub.inj.sup.2 is the variance due to injection, .sigma..sub.ther.sup.2 is the variance due to the thermal effects in the separation process, and .sigma..sub.a.sup.2 is the variance of the peak due to all other factors.
Current gel drying methods involve conventional oven drying. This method necessarily involves removing the gel from the electrophoresis apparatus and placing the gel into an oven.
When gel electrophoresis is conducted in a gel electrophoresis apparatus having an air impingement subassembly, gel drying can be accomplished using the same apparatus if desired with minor modifications, such as removing one of the glass plates from the gel and removing the electrophoresis buffer.
To summarize, the present invention results in improved resolution in less time.